Nanobodies are poised to provide a next wave of antibody based reagents for immunotherapy and in vivo medical imaging. However efficient methods of nanobody delivery with localized functional expression are still in their infancy. Neural stem cells (NSC) expressing therapeutic proteins have emerged as one of the most promising cell based therapeutic approaches to eradicate gliomas. In prior research, we have employed dual bioluminescence imaging and intravital microscopy and established that: a) NSC migrate extensively to sites of cerebral pathology;and b) engineered secreted therapeutic, S-TRAIL selectively induces apoptosis in proliferating glioma cells. In close collaboration with: a) Henegouwen lab (Utrecht University, The Netherlands) which is leading efforts in developing low molecular weight, highly soluble EGFR specific nanobodies (antibodies consisting of only single immunoglobulin (Ig) variable region);and b) Kim lab (McLean Hospital, Boston) which has successfully employed embryonic stem (ES) derived neural stem cells (eNSC) in mouse models of neuro-degeneration, we will initially develop novel neural stem cells expressing EGFR specific imageable and therapeutic nanobodies for their on-site delivery: a) to block EGF induced cell signaling in gliomas;and b) to follow their tumor targeting properties and fate in real time. Recent reports describe a strong synergistic tumoricidal effect for strategies in which EGFR signaling antagonists are combined with cytokine TRAIL, that is known to induce apoptosis by binding to death domain-containing receptors, DR4 and DR5 specifically present in tumor cells. Based on this hypothesis, we will also develop neural stem cells expressing secretable EGFR nanobodies and S-TRAIL and test the efficacy of combination therapies in mouse glioma models. The integration of genetically engineered fluorescent and bioluminescent imaging markers and in vivo imaging in close collaboration with Weissleder lab (MGH, Boston) and the other imaging groups in our center (CMIR- MGH, Boston) will allow us to follow delivery of NSC and to asses the therapeutic efficacy of engineered NSC and thus to adjust and fine tune the proposed therapeutic approaches. The developed agents and strategies will be designed to be clinically translatable and should have a major impact in developing efficient therapies for brain tumors. Nanobodies are poised to provide a next wave of antibody based reagents for immunotherapy and in vivo medical imaging. The overall goal of this proposal is to develop novel imageable and therapeutic recombinant nanobody secreting neural stem cells for on-site delivery of therapeutic agents in an effort to simultaneously target multiple components essential to brain tumor growth and survival. The developed agents and strategies will be designed to be clinically translatable and should have a major impact in developing efficient therapies for brain tumors.